Genes provide cells with the instructions for making proteins, and when a gene is silenced, the cell stops making the protein specified by that gene. RNA interference is a natural mechanism for silencing specific genes. RNA interference was first observed in plants. However, the first crucial breakthrough in understanding the RNAi mechanism came from studies of worms in 1998. The study recognized that double-stranded RNA (dsRNA) played a pivotal role in RNAi. In 2006, two American scientists, Andrew Fire and Craig C. Mello, shared the nobel prize in physiology for their work on RNA interference in the nematode worm Caenorhabditis elegans.
The induction of RNA interference using dsRNA has become a powerful tool for researchers to study the function of genes in many lower organisms, including worms and fruit flies. However, this approach initially seemed unworkable in mammalian cells, due to dsRNA tendency to provoke the interferon response and cause cell suicide. Such cell suicide makes biological sense in the normal situation where dsRNA is encountered - viral infection - because it prevents replication and spread of the virus to neighboring cells.
Success stories
Macular degeneration: The first RNAi therapy to reach patients in clinical trials would aim at a debilitating eye disease called macular degeneration. Most critically, RNAi drugs can be delivered directly to the diseased tissue, literally injected into the eye. This direct delivery ensure that 'naked' RNAi drugs, short strands of RNA that aren't packaged and protected in membranes and which quickly break down in the bloodstream, can reach their target intact. Local delivery reduces the chances of unanticipated, harmful effects of drugs elsewhere in the body.
Also, the disease is triggered by a protein called VEGF. This protein promotes blood vessel growth. In patients with macular degeneration, surplus of VEGF leads to sprouting of excess blood vessels behind the retina. The blood vessels leak, often entirely destroying vision. The new RNAi drugs shut down genes that produce VEGF and allow it to make the leaky vessels.
The first clinical trial, involving about two dozen patients, has launched in the fall of 2004. Two months after being injected with the drug, a quarter of the patients had significantly clearer vision, and the other patients' vision had at least stabilized. If subsequent trials prove they are effective, RNAi drugs for this condition could hit the market by 2009.
Hepatitis C: In 2002, Anton McCaffrey and Mark Kay at Stanford University announced that their RNAi treatment had controlled the virus in laboratory mice. It was for the first time an RNAi approach had worked not just in lab cell cultures but also in living animals. They injected naked RNA strands into the tail veins of mice using a high-pressure, rapid-transfusion method to ensure that the RNA strands were taken up in the liver. But the delivery method used, which doubled the mice's blood volume within eight seconds, isn't feasible for humans. And even if it were, the effects of naked RNA are likely to wear off in a matter of days. So these researchers are finding ways to use viral vectors, viruses stripped of their harmful genes, to carry RNA-making molecules into liver cells.
Huntington's disease: In 2004, Beverly Davidson and colleagues at the University of Iowa used this technique to treat mice with spinocerebellar ataxia, a neurological disorder akin to huntington's. It was a landmark-the first time that an actively troublesome gene was put out of commission in such a way. Soon after, Davidson treated mice with huntington's, a disease that affects more than 30,000 people in the U.S. alone. But, in addition to silencing the harmful gene, the treatment also shut down the healthy version of the Huntington's gene (Patients carry both).
HIV: In 2002, Phillip Sharp and colleagues at MIT announced they could interrupt various steps in the HIV life cycle with RNAi molecules. But these and other experiments were largely 'proof-of-principle' studies, stopping the virus in cell cultures, not in human patients.
HIV mutates and evolves resistance so rapidly that any single target for an RNAi therapy won't be sufficient. Molecular biologist John Rossi of City of Hope Medical Center and colleagues at Colorado State University have engineered an RNAi therapy aiming at multiple HIV genes. And to build up the multipronged attack even more, Rossi combines this RNAi therapy with two other RNA technologies (called ribozymes and RNA decoys) to block HIV's replication and invasion of the immune system.
Respiratory infections: It's relatively simple to deliver RNAi drugs directly to the respiratory system, as patients can inhale them. Yes, one day, we may breathe in RNAi drugs for a host of different viruses, including SARS and influenza. The first virus to be defeated in this way, though, will likely be respiratory syncytial virus, or RSV.
By early 2005, biochemist Sailen Barik at the University of South Alabama had engineered RNAi molecules to shut down various RSV genes. Like the treatment for macular degeneration, these molecules were short strands of naked RNA that would rapidly break down in the bloodstream. When inhaled by mice, though, the short RNA strands reached their targets intact and controlled the virus. Clinical trials for RSV are slated to begin in the first half of 2006.
Cancer: In the last few years, researchers have silenced more than a dozen known cancer-causing genes with RNAi. Yet, once again, most of this success has been with cell cultures in the lab and delivery poses the key hurdle in moving from the lab to the bedside of patients. Researchers are just beginning to sort how RNAi therapies might reach and penetrate tumors.
Rather than take a leading role, some RNAi therapies may help defeat cancers by supporting chemotherapy. Drug resistance is a major problem in chemotherapy. In many of these failures, the guilty agent is a protein called P-glycoprotein, which sweeps drugs out of diseased cells. In 2004, a team of scientists at Imperial College London showed that RNAi could stop production of the protein in multidrug-resistant leukemia cells, restoring their sensitivity to existing drugs.
RNAi for Big Pharma
RNA interference (RNAi) has revolutionized not only the basic field of molecular biology, but also its applications in pharmaceutical research, in which RNAi can be used both to identify drug targets and to treat diseases.
As genes are important targets for the development of drugs, the pharmaceutical industry is highly interested in RNAi technology. RNAi-based drugs have the potential to be more selective and hence more effective and less toxic than traditional drugs. Some people even envisage the creation of a new class of drugs on the condition that further progress is made in RNAi therapy. Three US American companies are testing the effect of RNAi fragments in clinical trials. They are investigating whether these fragments might be able to stop the proliferation of blood vessels in the eyes (macular degeneration). RNAi, along with other related technologies, will contribute to the development of personalized medicine.
Although none of the RNAi-based drugs is in the market yet, some are in clinical trials. By the year 2010 the market for RNAi-based drugs is expected to be worth $3.5 billion and is expected to expand to $10.5 billion by the year 2015.
Despite the proliferation of promising cell culture studies for RNAi-based drugs, some concern has been raised regarding the safety of RNA interference, especially the potential for 'off-target' effects in which a gene with a coincidentally similar sequence to the targeted gene is also repressed. A computational genomics study estimated that the error rate of off-target interactions is about 10 per cent. One major study of liver disease in mice led to high death rates in the experimental animals, suggested by researchers to be the result of 'over saturation' of the dsRNA pathway.
The promise of RNAi
When RNAi emerged on the research scene five years ago, from experiments at Whitehead and other labs, it was hailed as a critical breakthrough for science. The approach involves delivering tiny strands of RNA into target cells. These strands interfere with the messenger RNA molecules that control protein production and hence gene expression, giving scientists the power to knock out individual genes at will.
RNAi promises to create new drugs that would target the genetic roots of disease. Many classes of RNAi-based drugs are at advanced stages of development. A treatment for age-related macular degeneration (AMD) of the eye is in phase 1 clinical trial. Other RNAi-based drugs still in pre-clinical development target HIV, hepatitis C, Huntington disease, and various neurodegenerative disorders.
In the race toward the clinic, RNA is at a turning point. While scientists generally remain upbeat about its clinical potential, they are still cautious of their views. Getting the tiny strands of RNA (known as short interfering RNAs) into target cells is not an easy task. The strong appeal of this technique is its potency and specificity, while its greatest challenge is the delivery of RNAi-based drugs, a process, which is still in the nascent stages of research.
(The author is with Accure Labs Pvt. Ltd., New Delhi)